Characterisation and partial purification of the GABA(B) receptor from the rat cerebellum using the novel antagonist [3H]CGP 62349

Brain Res Mol Brain Res. 1999 Aug 25;71(2):279-89. doi: 10.1016/s0169-328x(99)00199-0.

Abstract

The novel GABA(B) receptor antagonist [3H]CGP 62349 binds rat cerebellar synaptosomal membranes with high affinity at a single population of sites (K(d) = 0.9 nM, B(max) = 760 fmol/mg protein). Solubilisation with 1% Triton X-100/0.5 M NaCl/10% glycerol resulted in a marked increase in [3H]CGP 62349 binding (K(d) = 0.5 nM, B(max) = 1285 fmol/mg protein). Competition of [3HCGP 35348 = CGP 36742. The GABA(A) ligand isoguvacine did not displace [3H]CGP 62349 binding. Partial purification of [3H]CGP 62349 binding sites was obtained by sucrose density centrifugation and a predominant protein in the peak binding fraction was recognised by an anti-GABA(B) receptor antibody and had a molecular weight similar to the recombinant expressed GABA(B)R1a. These results demonstrate that [3H]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABA(B) receptor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzoates / metabolism
  • Benzoates / pharmacology*
  • Binding Sites
  • Biotinylation
  • COS Cells
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Cerebellum / chemistry*
  • Cerebellum / drug effects
  • Detergents / pharmacology
  • Electrophoresis, Polyacrylamide Gel
  • GABA-B Receptor Antagonists
  • Isonicotinic Acids / metabolism
  • Kinetics
  • Organophosphorus Compounds / metabolism
  • Organophosphorus Compounds / pharmacology*
  • Rats
  • Receptors, GABA-B / isolation & purification*
  • Transfection

Substances

  • Benzoates
  • CGP 62349
  • Detergents
  • GABA-B Receptor Antagonists
  • Isonicotinic Acids
  • Organophosphorus Compounds
  • Receptors, GABA-B
  • isoguvacine